MODELING THE 3D STRUCTURE OF OPIOID RECEPTORS

点击翻译按钮获取中文摘要

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. The primary goal of the research proposed here is to further develop and apply a new approach for the construction of reliable three dimensional models of the delta, mu, and kappa opioid receptors as typical examples of the rhodopsin-like family of G-protein coupled receptors (GPCR). The approach assumes spatial proximity for residues with similar conservation patterns and posits that all polar sidechains within the transmembrane core will participate in intramolecular hydrogen bonding. In the first stage of the modeling, the sequential assignment of 7 GPCR transmembrane alpha-helices to the peaks of the 9 Angstroms resolution electron microscopy projection map of bovine rhodopsin can be deduced from mutagenesis and cross-linking data and from analysis of helix inner arcs occupied by evolutionary conserved and hydrophilic residues in 5 sequence alignments of 427 GPCRs. Then, the "maximum hydrogen bonding participation" criterion and the inferred spatial proximity of residues conserved in a correlated fashion in sequence alignments of GPCR are used to find rotational orientations of the alpha-helices, to align the helices in the direction perpendicular to the membrane plane, and to derive constraints for refinement of the obtained receptor structure with distance geometry calculations. Although only opioid receptors are initially targeted, the approach is generally applicable for all members of the large (>480 unique sequences) rhodopsin-like subset of the GPCR superfamily. In preliminary studies we have constructed an approximate model of the transmembrane core of the delta opioid receptor and calculated a preliminary set of its conformations (with coordinate r.m.s.d. of Calpha atoms about 1.5 Angstroms) using distance constraints.derived from analysis of the sequence alignment of 105 peptide<GPCR. The conformations with lowest target function provide hydrogen bonding for all intramembrane polar side chains, spatial proximity of residues with similar conservation patterns, and compact packing of alpha-helices. In this application, we propose to: 1. Refine our preliminary model of the delta opioid receptor by reanalysis of distance constraints and further calculations with the distance geometry algorithm and energy minimization; 2. investigate preliminary evidence of distinct hydrogen bonding networks in proposed active and inactive conformations of the delta receptor; 3. Extend the modeling approach to include the loop regions, linking the transmembrane helices; 4. Use our recently described model of the binding conformations of delta receptor agonists and antagonists in conjunction with the receptor structure to model ligand- delta receptor recognition; 5. Extend the analysis of the delta receptor to mu and kappa opioid receptors; 6. Use the insights gained from the receptor-ligand modeling to design new ligands with specific pharmacological attributes.